Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. 1. The Field of the Invention
This invention relates to the medical arts. In particular the present invention relates to the field of cellular signal transduction and to gene therapy.
2. Discussion of the Rekated Art
Cytokines are small secreted proteins or factors (5 to 20 kD) that have specific effects on cell-to-cell interactions, intercellular communication, or the behavior of other cells. Cytokines involved in inflammatory diseases are produced by lymphocytes, especially TH1 and TH2 lymphocytes, monocytes, intestinal macrophages, granulocytes, epithelial cells, and fibroblasts. (Reviewed in G. Rogler and T. Andus, Cytokines in inflammatory bowel disease, World J. Surg. 22(4):382-89 [1998]; H. F. Galley and N. R. Webster, The immuno-inflammatory cascade, Br. J. Anaesth. 77:11-16 [1996]). Some cytokines are pro-inflammatory (e.g., tumor necrosis factor [TNF]-xcex1, interleukin [IL]-1 (xcex1and xcex2), IL-6, IL-8, IL-12); others are anti-inflammatory (e.g., IL-1 receptor antagonist [IL-1ra], IL-4, IL-10, IL-11, and transforming growth factor [TGF]-xcex2). However, there may be overlap and functional redundancy in their effects under certain inflammatory conditions.
One group of cytokines, the IL-6-type, are also important in the regulation of complex cellular processes such as gene activation, proliferation and differentiation. The IL-6-type cytokines include IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neutrophic factor, and cardiotrophin-1. (Reviewed in P. C. Heinrich et al., Interleukin-6-type cytokine signaling through the gp130/JAK/STAT pathway, Biochem. J. 334(Pt 2):297-314 [1998]). The IL-6-type cytokines (also known as the gp130 signaling subunit cytokine family) have in common that signal transduction proceeds through a pathway beginning with ligand binding by type I and type II surface receptors, internalization involving affinity converter/signal transducing subunit gp130, the activation of the Janus family of cytoplasmic tyrosine kinases (e.g., Jak1, Jak2, and Tyk2); this results in the phosphorylation and dimerization of the signal transducers and activators of transcription (STAT)-1 and STAT-3 that activate transcription from promoters having STAT recognition sites. (Heinrich et al. [1998]; M. Ernst et al., Gp130-mediated signal transduction in embryonic stem cells involves activation of Jak and Ras/mitogen-activated protein kinase pathways, J. Biol. Chem. 271(47):30136-43 [1996]; R. Starr et al., A family of cytokine-inducible inhibitors of signaling, Nature 387(6636):917-21 [1997]; T. Hirano et al., Cytokine and Growth Factor Rev. 8:241-52 [1997]; E. Arzt and G. K. Stalla, Neuroimmunomodulation 3:28-34 [1996]; S. J. Haque and B. R. G. Williams, Semin. Oncol. 25 (suppl. 1):14-22 [1998]). This pathway is known as the Jak-STAT signaling cascade.
Several IL-6-type cytokines are important neuro-immuno-endocrine modulators of the hypothalamo-pituitary-adrenal (HPA) axis (Arzt, E. and Stalla [1996]; S. Melmed, Trends Endocrinol. Metab. 8:391-97 [1997]; H. O. Besedovsky, and A. Del Rey, Endo. Rev. 17:64-102 [1996]), which regulates metabolism, including growth, body temperature, water balance, blood sugar, fat metabolism, and sexual and nerve function. For example, LIF is a potent auto-paracrine stimulus of pituitary proopiomelanocortin (POMC) gene expression and adrenocorticotrophic hormone (ACTH) secretion, which stimulates the adrenals to produce additional hormones. Thus, LIF modulates the HPA axis response to various inflammatory and stress stimuli. (Z. Wang et al., Endocrinology 137:2947-53 [1996]; C. J. Auernhammer et al., Endocrinology 139:2201-08 [1998a]). In vitro experiments using human fetal pituitary cells (I. Shimon et al., J. Clin. Invest. 100: 357-63 [1997]) and the corticotroph cell line AtT-20 (S. Akita et al., J. Clin., Invest 95, 1288-1298 [1995]; C. Bousquet et al., J. Biol. Chem 272:10551-57 [1997]), showed a profound and synergistic action of LIF and corticotropin-releasing hormone (CRH) on POMC gene expression and ACTH secretion. LIF is known to induce the Jak-STAT signaling cascade in the corticotroph cells. (C. J. Auernhammer et al., Pituitary corticotroph SOCS-3: novel intracellular regulation of leukemia-inhibitory factor-mediated proopiomelanocortin gene expression and adrenocorticotropin secretion, Mol. Endocrinol. 12(7):954-61 [1998b]; I. Shimon et al. [1997]; D. W. Ray et al., Leukemia inhibitory factor (LIF) stimulates proopiomelanocortin (POMC) expression in a corticotroph cell line. Role of STAT pathway, J. Clin. Invest. 97(8):1852-59 [1996]; D. W. Ray et al., Ann. N.Y. Acad. Sci. USA 840:162-73 [1998]).
A new family of cytokine-inducible proteins has recently been described that inhibits the Jak-STAT signaling cascade. (E.g., S. E. Nicholson et al., The SOCS proteins: a new family of negative regulators of signal transduction, J. Leukoc. Biol. 63(6):665-68 [1998]; R. Starr et al., SOCS: suppressors of cytokine signaling, Int. J. Biochem. Cell. Biol. 30(10):1081-85 [1998]). These proteins have been variously termed suppressors of cytokine signaling (xe2x80x9cSOCSxe2x80x9d)(R. Starr et al., A family of cytokine-inducible inhibitors of signaling, Nature 387(6636):917-21 [1998]; D. J. Hilton et al., Proc. Natl. Acad. Sci. USA 95:114-19 [1998]), STAT-induced STAT inhibitors (SSI)(T. Naka et al., Nature 387:924-28 [1997]; S. Minamoto et al., Biochem. Biophys. Res. Commun 237:79-83 [1997]), cytokine-inducible SH2 containing protein (CIS)(A. Yoshimura et al., EMBO J. 14:2816-26 [1995]; M. Masuhara et al., Biochem. Biophys. Res,. Commun. 239:439-46 [1997]; A, Matsumoto et al., Blood 89:3148-54 [1997]), and Jak binding protein (JAB)(T. A. Endo et al., Nature 387:921-24 [1997]; H. Sakamoto et al., Blood 92:1668-76 [1998]). The SOCS-protein family currently consists of CIS and SOCS-1 through 7. (D. J. Hilton et al. [1998]; M. J. Aman and W. J. Leonard, Curr. Biol. 7:R784-R788 [1997]; R. Starr and D. J. Hilton, Int. J. Biochem. Cell Biol. 30:1081-85 [1998]).
SOCS-protein expression is stimulated by various cytokines in a tissue specific manner (R. Starr et al., Nature 387:917-21 [1997]; M. J. Aman and W. J. Leonard [1997]; H. Sakamoto et al. [1998]; H. O. Besedovsky, and A. Del Rey [1996]; T. E. Adams et al., J. Biol. Chem. 273:1285-87 [1998]; C. Bjorbaek et al., Mol. Cell 1:619-625 [1998]). The gene expression of SOCS-1/SSI-1/JAB and SOCS-3/SSI-3/CIS-3, referred to herein as SOCS-1 and SOCS-3, are induced by IL-6 and LIF in various tissues (R. Starr et al. [1997]; D. J. Hilton et al. [1998]; T. Naka et al. [1997]; S. Minamoto et al. [1997]; M. Masuhara et al. [1997]; A. Matsumoto et al. [1997]; T. A. Endo et al. [1997]). For example, SOCS-3 gene expression is rapidly induced by LIF in the pituitary in vivo, and in corticotroph AtT-20 cells in vitro. (C. J. Auernhammer et al. [1998b]).
Both, SOCS-1 and SOCS-3 proteins bind to the JH1 domain of Jak-2 and thereby inhibit IL-6-, IL-11-, or LIF-induced tyrosine phosphorylation activity by Jak-2 of gp130 and STAT-3. (S. Minamoto et al. [1997]; M. Masuhara et al. [1997]; C. J. Auernhammer et al. [1998b]). SOCS-3 is induced by growth hormone (GH) in the liver, and inhibits GH-induced Spi 2.1 promoter activity. (T. E. Adams et al. [1998]). SOCS-3 inhibits LIF-induced POMC gene expression and ACTH secretion (C. J. Auernhammer et al. [1998b]), thus providing an intracellular negative feedback regulation of cytokine-induced activation of the HPA-axis. Hypothalamic SOCS-3 gene expression is stimulated by leptin, and SOCS-3 inhibits leptin-induced signal transduction (C. Bjorbaek et al., Mol. Cell 1:619-625 [1998]), thus suggesting its regulatory role in central leptin resistance.
The structure of SOCS proteins has been described. (e.g., S. E. Nicholson et al., Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction, EMBO J. 18(2):375-85 (January 1999). Dominant negative STAT-3 mutants, isolated by substitution of a carboxy-terminal tyrosine phosphorylation site Tyr705 to Phe705 (STAT-3F) or mutation at positions important for DNA binding (STAT-3D) have been recently described (K. Nakajima et al., EMBO J. 15:3651-58 [1996]). Overexpression of these STAT-3 dominant negative mutants in corticotroph AtT-20 cells inhibits LIF-induced POMC gene expression and ACTH secretion. (C. Bousquet and S. Melmed, J. Biol. Chem. 274:10723-30 [1999]). Cytokine-induced gene expression of SOCS-1 has been shown to be inhibited in cells overexpressing dominant negative STAT-3 mutants (T. Naka et al. [1997]), but the promoter region of SOCS-1 has not been cloned.
Therefore, there remains a definite need for a promoter sequence capable of regulating expression of preselected proteins, such as SOCS-3 protein, and that can be targeted by gene therapy to treat growth disorders, autoimmune diseases, immune diseases, and inflammatory conditions. This and other features and benefits provided by the present invention will now be described.
The present invention relates to a nucleic acid construct comprising a murine SOCS-3 promoter sequence, or a non-murine homologue thereof, or an operative fragment or derivative of any of these. The construct can also contain, operatively linked to the SOCS-3 promoter, a DNA sequence encoding a gene for any preselected protein or a gene-specific part of such a DNA sequence, or to a DNA sequence that encodes a preselected gene-specific antisense RNA or a catalytic RNA. A preselected protein that is encoded by the nucleic acid construct can be from an autologous, allogeneic, or xenogeneic source. In addition, the present nucleic acid construct optionally contains a reporter gene to facilitate detection and/or selection of successfully transfected cells. The present nucleic acid construct is particularly useful for linking expression of a desired gene product to physiological processes that are regulated by gp130-mediated signal transduction from IL-6-type cytokines (i.e., cytokines of the gp130 signaling subunit cytokine family), such as IL-6, IL-11, or LIF. For example, when the encoded protein is a SOCS-3 protein, the present nucleic acid can be used to modulate the physiology and/or hormonal secretions of cells of the hypothalamus, pituitary, adrenals, liver, or other tissues, through a negative autoregulatory feedback of SOCS-3 on its own cytokine-induced gene expression.
The present invention also relates to a transgenic vertebrate cell containing the nucleic acid construct of the present invention and to transgenic non-human vertebrates comprising such cells.
The present invention also relates to a method of treating a growth retardation disorder in a human subject. The method involves genetically modifying a GH-responsive or gp130-responsive cell(s) of a human subject having a growth retardation disorder, such as dwarfism, GH deficiency, gonadal dysgenesis, chondrodystrophy, or bone-cartilage dysplasia. The cell(s) are genetically modified using a nucleic acid construct that comprises a SOCS-3 promoter sequence, or operative fragment thereof, operatively linked to a DNA sequence that encodes an RNA that specifically hybridizes to a functional SOCS-3 mRNA. In response to a growth-inducing cytokine, in vivo, the genetically modified cell(s) within the human subject, transcribe an RNA transcript that specifically hybridizes to a functional SOCS-3 mRNA, preventing translation therefrom. This RNA transcript can be an antisense RNA or a catalytic RNA (ribozyme) that cleave the SOCS-3 mRNA. As a consequence, the amount of SOCS-3 protein produced within the genetically modified cell(s) is relatively reduced, and one or more symptoms of the growth retardation disorder in the subject are thereby improved, due to a lessening of SOCS-3-mediated signal suppression within the genetically modified cell(s).
The present invention also relates to a method of treating a growth acceleration disorder in a human subject. The method involves genetically modifying a GH-responsive or gp130-responsive cell(s) of a human subject having a growth acceleration disorder, such as gigantism, acromegaly, or Cushing""s disease. The cell(s) are genetically modified using a nucleic acid construct, comprising a SOCS-3 promoter sequence, or operative fragment thereof, operatively linked to a DNA sequence encoding a SOCS-3 protein, or functional fragment thereof. In response to the growth-inducing cytokine, in vivo, the genetically modified cell(s) produce an enhanced amount of SOCS-3 protein. The symptom(s) of the growth acceleration disorder in the subject are thereby improved, due to enhanced SOCS-3-mediated cytokine signal suppression.
The present invention also relates to a method of treating an autoimmune disease, immune disease, or inflammatory condition in a human subject having a condition, such as Crohn""s disease, ulcerative colitis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Grave""s disease, or a neuroendocrinological response to psychological or physical stress. The method involves genetically modifying a gp130-responsive cell(s), responsive to a pro-inflammatory cytokine, such as IL-6 or LIF. The cell(s) are genetically modified using a nucleic acid construct that includes a SOCS-3 promoter sequence, or operative fragment thereof, operatively linked to a DNA sequence encoding a SOCS-3 protein, or functional fragment thereof. In response to a pro-inflammatory cytokine of the gp130 signaling subunit cytokine family, in vivo, the genetically modified cell(s) produce an enhanced amount of SOCS-3 protein. The symptom(s) of the autoimmune disease, immune disease, or inflammatory condition in the subject are thereby improved, due to a relative increase in SOCS-3-mediated signal suppression.
Alternatively, the SOCS-3 promoter is operatively linked to a DNA sequence encoding a functional anti-inflammatory cytokine of the gp130 signaling subunit cytokine family, such as IL-11, linked to a functional secretory signal. In response to a pro-inflammatory cytokine of the gp130 signaling subunit cytokine family, in vivo, the genetically modified cell(s) produce and secrete an enhanced amount of the anti-inflammatory cytokine. The symptom(s) of the autoimmune disease, immune disease, or inflammatory condition in the subject are thereby improved.
The present invention also relates to a kit for genetically modifying a vertebrate cell. The kit includes a polynucleotide comprising a murine SOCS-3 promoter sequence having SEQ. ID. NO.:1, or an operative fragment or non-murine homologue thereof, or an operative derivative of any of these. Preferably, the polynucleotide includes, operatively linked to the SOCS-3 promoter, at least one DNA sequence encoding a preselected protein or a gene-specific part of such a DNA sequence, or a DNA encoding a preselected gene-specific antisense RNA or a specific catalytic RNA, as appropriate for a particular application. Optionally, the promoter is linked to a reporter gene for facilitating detection, isolation, or selection of genetically modified cells from unmodified cells. Some embodiments of the kit are configured for use in practicing the present methods of treating a growth retardation or acceleration disorder in a human subject or the present method of treating an autoimmune disease, immune disease, or inflammatory condition in a human subject.
These and other advantages and features of the present invention will be described more fully in a detailed description of the preferred embodiments which follows.